Difference between revisions of "Part:BBa K2332317"
 
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Latest revision as of 00:32, 23 October 2017


Photoactive dCas9-p65 (siLACE system)

siLACE
Function Photo-sensitive transcription activation
Use in Mammalian cells
Abstraction Hierarchy Part
RFC standard RFC10, RFC12, RFC23 & RFC25 compatible
Backbone pSB1C3
Submitted by [http://2017.igem.org/Team:UCL UCL iGEM 2017]

This gene encodes LIT's single-illumination light-activated CRISPR-Cas9 effector (siLACE) system. It is fusion of the 'plasma membrane delivery system' BioBrick BBa_K2332315 and the 'dCas9-p65 (TF)' BioBrick BBa_K2332316.

The siLACE system allows activation of endogenous and exogenous genes of interests with a single laser light exposure of the cell.


Usage and Biology

...

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 6712
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 704
    Illegal BsaI.rc site found at 1471


Functional Parameters

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)
 ACCATGCGGGCC ... AGCGGATCCTAG
 ORF from nucleotide position 4 to 6717 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
1 
101 
201 
301 
401 
501 
601 
701 
801 
901 
1001 
1101 
1201 
1301 
1401 
1501 
1601 
1701 
1801 
1901 
2001 
2101 
2201 		MRAWIFFLLCLAGRALAATGRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQH
DFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADH
YQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKDYKDDDDKTGGGGGSGGGGSPSIATGMVGALLLLLVVALGIGL
FMRRRHIVRKRGGGGSGGGGSGSVIPDYFKQSFPEGYSWERSMTYEDGGICIATNDITMEGDSFINKIHFKGTNFPPNGPVMQKRTVGWEASTEKMYERD
GVLKGDVKMKLLLKGGGHYRCDYRTTYKVKQKPVKLPDYHFVDHRIEILSHDKDYNKVKLYEHAVARNSTDSMDELYKGGSGGMVSKGEETITSVIKPDM
KNKLRMEGNVNGHAFVIEGEGSGKPFEGIQTIDLEVKEGAPLPFAYDILTTAFHYGNRVFTKYPRGGGGTGGGGSGGGGSDKKYSIGLAIGTNSVGWAVI
TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN
IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRR
LENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM
IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA
ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY
NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL
TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH
EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRD
MYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL
VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM
IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA
RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ
KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP
AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDEGAPKKKRKVGSSGSPKKKRKVGSQYLPDTDDRHRIEEKRKRTYETFKSIMKK
SPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAP
GPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAP
APLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISSGS*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
RFC25 scar (shown in bold): 19 to 20, 266 to 267
Flag-tag: 258 to 265
Enterokinase cleavage site: 261 to 265
SV40 nuclear localization sequence: 1951 to 1957, 1963 to 1969
Amino acid composition:
Ala (A)133 (5.9%)
Arg (R)113 (5.0%)
Asn (N)98 (4.4%)
Asp (D)153 (6.8%)
Cys (C)7 (0.3%)
Gln (Q)80 (3.6%)
Glu (E)155 (6.9%)
Gly (G)171 (7.6%)
His (H)52 (2.3%)
Ile (I)132 (5.9%)
Leu (L)214 (9.6%)
Lys (K)212 (9.5%)
Met (M)44 (2.0%)
Phe (F)100 (4.5%)
Pro (P)105 (4.7%)
Ser (S)134 (6.0%)
Thr (T)114 (5.1%)
Trp (W)11 (0.5%)
Tyr (Y)86 (3.8%)
Val (V)124 (5.5%)
Amino acid counting
Total number:2238
Positively charged (Arg+Lys):325 (14.5%)
Negatively charged (Asp+Glu):308 (13.8%)
Aromatic (Phe+His+Try+Tyr):249 (11.1%)
Biochemical parameters
Atomic composition:C11297H17862N3082O3367S51
Molecular mass [Da]:252365.8
Theoretical pI:8.69
Extinction coefficient at 280 nm [M-1 cm-1]:188640 / 189078 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges 
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):good (0.76)good (0.77)good (0.74)good (0.78)good (0.76)very bad (0.00)
Alignments (obtained from PredictProtein.org)
   There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.
Predictions (obtained from PredictProtein.org)
   There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation.
If you have any questions, comments or suggestions, please leave us a comment.


Reference

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